Methylation is a epigenetic state occurring in some portions of most genomes. It can have significant effects on gene expression and has been correlated with the etiology of cancer and common disease. The BeadXpress system from Illumina is capable of efficiently assaying differential methylation profiles at 48 to ~1500 independent loci. The assay enables very sensitive and specific interrogation of genomic regions.

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How Does This Work?

The Basics

The methylation assay performed on the BeadXpress comprises several steps.

First, the gDNA of interest is used in a bisulfate conversion reaction which causes the cytosine nucleotides to become uracil (C->U). Regions of the genome that are methylated contain only 5-methylcytosine instead of normal cytosine and are protected from the conversion. Thus, in methylated regions, the cytosine nucleotides remain cytosine, while those in unmethylated regions become uracil.

At this point, the Illumina Golden Gate assay enables us to design oligos to very specifically amplify the sequence around any particular location and label the Cytosines and Uracils within the amplified alleles with different fluorescent dyes (Cy3 and Cy5). We can include up to 384 loci in one reaction well for each DNA sample, a process called “multiplexing”.

Then, these amplified alleles are hybridized to and immobilized on a special substrate built by Illumina, called a VeraCode bead. From here, the Illumina BeadXpress machine can automatically determine the identity of oligo primer hybridized to the VeraCode beads, and measure and quantify the fluorescent signal (thus indicating the genotype) for each amplified locus.

The presence of the fluorescent dye bound to cytosine is indicative of methylation. A mixture of the two fluorescent dyes can indicate partial methylation, and the absence of cytosine signal dye indicates an unmethylated state in that region.

For a more detailed description of the mechanics and chemistry, we recommend that you consult the Illumina web site.

How to Get Started

Our clients have a choice in this. Either, they develop their target loci list and flanking oligos independently, or they choose to have our bioinformatics specialists assist them in this process.

Once an oligo list has been created for the loci of interest, this file is provided to Illumina as one part of the order. Illumina will use the SNP and oligo list to create an OMA (oligo methylation array) which is actually the list of which VeraCode beads will match to which SNP oligos.

Illumina then sends the OMA and associated reagents to our laboratory. Once you’ve sent your DNA, we perform the bisulfide conversion steps and initiate the Golden Gate assay using your OMA. When your data is generated and analyzed for accuracy, we send it to you.